The present invention relates to a kit for optically detecting proteins, to a method for optically detecting proteins, to a method of analysing proteins and to the use of polymethines for optically detecting proteins in a sample.
Determination of the circulating levels of plasma lipoproteins is important in the diagnosis of primary and secondary disorders of lipid transport and in risk assessment for arteriosclerosis and coronary artery disease. In the fasting state, three main lipoprotein classes have been identified: VLDL (very low density lipoproteins), LDL (low density lipoproteins) and HDL (high density lipoproteins), each of which differs in size and density, and in lipid and apolipoprotein composition.
It is well established that there is a positive correlation between risk of premature coronary heart disease and total plasma cholesterol and plasma LDL-cholesterol (LDL-C). There is also a correlation between decreased HDL-cholesterol (HDL-C) and increased plasma TG (triglycerides). Heterogeneity in the size and density of LDL is well documented and has also been shown to have clinical relevance. Small dense LDL (Pattern B) has an increased relative risk compared with large light LDL (Pattern A). One of the most prevalent lipid/lipoprotein patterns associated with risk of coronary artery disease is the atherogenic lipoprotein phenotype (ALP), which is characterized by moderately raised plasma TG, low levels of HDL-C, elevated total and LDL-C, and small, dense LDL particles. Although methods are available in the clinical laboratory for measurement of HDL, LDL and VLDL, methods for the identification of the predominant LDL subclass are technically difficult and time-consuming.
The main methods for separation and analysis of the plasma lipoprotein levels, based on differences in physical properties, include ultracentrifugation, electrophoresis and differential precipitation. Of these, ultracentrifugation is seen in the prior art as the “gold standard” for analysis of plasma lipoproteins and potentially provides the greatest amount of information, as the lipid and apolipoprotein compositions of the separated lipoproteins can be analysed.
Methods for density gradient centrifugation have generally been based on salt solutions, and include sequential flotation with adjustment of the density of the plasma and infranatants after each centrifugation step, or centrifugation on discontinuous or continuous gradients. The use of salt gradients has a number of disadvantages. These are technically difficult to prepare and relatively unstable, and reproducibility is difficult to achieve. In addition, prolonged centrifugation is often necessary to float the lipoproteins into the gradients and the high salt concentrations can modify the protein structure and lead to loss of apolipoproteins from the lipoprotein fractions. For further analysis of the lipoprotein fractions, it is usually necessary to remove the salt, e.g., by dialysis. This results in loss of material and poor recoveries.
Hence, it would be desirable to provide improved methods for analysing lipoproteins and in particular for characterizing sub-class patterns of the lipoproteins in order to better characterize the atherogenic risk of a patient and to obtain more information for managing patients.